CN103232951B - Enterobacter gergoviae and its use in bioflocculation - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and specifically relates to a method for preparing a biological flocculant by Enterobacteriaceae and its use, specifically the application of Enterobacter diazoae in the preparation of a microbial flocculant, and its preparation method as a microbial flocculant is to inoculate the strain Into the sterilized seed medium, after cultivation, take the seed solution and inoculate it into the fermentation medium, cultivate it at 30°C for 18 hours, then centrifuge the fermentation liquid, and remove the supernatant to obtain the biological flocculant; the process of the present invention Simple and low cost; the prepared flocculant has high activity, strong heat resistance, good pH tolerance, and no secondary pollution. It can strengthen the settleability of sludge in sewage treatment, and the flocculation rate can reach 90%. application prospects.

Description

Enterobacteriaceae and its use in bioflocculation

technical field

The invention belongs to the technical field of microbes, and in particular relates to a method for preparing a biological flocculant by enterobacteria and an application thereof.

Background technique

my country is one of the countries with serious water shortage and pollution. In order to solve the problem of water shortage, in addition to saving water, strengthening the treatment of sewage is an urgent problem to be solved at present. Among water treatment methods, flocculation is one of the most commonly used methods. Domestic sewage and various industrial wastewater often contain different types and quantities of suspensions and colloids. By adding flocculants in water treatment, these sols and suspensions are destabilized, and then condensed into large particles and quickly precipitated.

There are many types of flocculants. Inorganic flocculants such as polyaluminum chloride and polyferric chloride are reliable in operation, but they are costly and have certain hazards. They are easy to eliminate one kind of pollution and bring another kind of pollution at the same time. The organic synthetic flocculant represented by polyacrylamide has a small dosage, fast speed and wide application range, but its residual monomers in water are not easy to be degraded, and it has a strong "three-to-one" effect. The application of natural polymer flocculant microbial flocculants has been reported in the 1960s, and commercial flocculants have been sold in the 1970s, such as Claron produced by Allyn Chemical Company and Moul-laracel produced by Moglu Company. Microbial flocculants can be divided into: (1) flocculants that directly use microbial cells; (2) flocculants that use microbial cell extracts; (3) flocculants that use microbial cell metabolites. Compared with common inorganic and organic synthetic flocculants, microorganisms have many unique advantages: (1) non-toxic and harmless, high safety, and can be used for post-fermentation treatment in food, pharmaceutical and other industries; (2) easy to be degraded , no secondary pollution; 3) It has a wide range of applications and unique decolorization effect. It has been used in the clarification of hydrometallurgy pulp, separation of inorganic suspensions, oil drilling, sewage decolorization, sludge dehydration, etc., after treatment with microbial flocculants The sewage is easier to separate solid and liquid, and the amount of sediment is less; (4) The pH value of some microbial flocculants is stable, the thermal stability is good, and the dosage is small; (5) The source is wide, the production cycle is short, and it can also be used Some wastewater is used as a culture medium to achieve the purpose of treating waste with waste.

The United States, Japan, and the United Kingdom are countries that carried out research on microbial flocculants earlier. Compared with foreign countries, my country started late, but has made great progress recently. There are also a large number of patents on microbial flocculants. For example, Zhao Xiaoxiang and Xia Lili applied for a composite microbial flocculant for cyanobacteria blooms, which can achieve 92% flocculation effect on cyanobacteria; Li Qiang applied for a radial sudden In the method for preparing biological flocculant by bacillus, the microbial flocculant has good heat resistance and has a treatment effect on sewage in the range of pH6-12; in the preparation method of a bacillus flocculant applied by He Huan, the microbial flocculant The flocculation effect of kaolin can reach more than 90%. The screening of high-efficiency flocculant-producing bacteria is still an important research content in microbial flocculants, and there is no report about Enterobacter as a flocculation microorganism.

Contents of the invention

In view of this, the present invention provides a new application of Enterobacter dilatatus, which provides a new idea for the treatment of sewage.

To achieve the above object, the technical solution of the present invention is:

Application of Enterobacter diazoae in the preparation of microbial flocculants.

The nucleotide sequence of the 16S rRNA of the Enterobacter diarrhoea strain is shown in SEQ ID NO:1.

Further, in the described application, the biological deposit number of the Enterobacter diarrhoea is CCTCC M2013042.

The second object of the present invention is to provide a preparation method of microbial flocculant and the product thereof, the method has low cost and stable process; the product has good pH value and temperature tolerance.

To achieve the above object, the technical solution of the present invention is:

The preparation method of the microbial flocculant specifically comprises: fermenting and cultivating the Enterobacter versicolor strain with a medium suitable for the Enterobacter versicolor, and separating, and the bacterial body or bacterial liquid is the microbial flocculant.

Further, the preparation method of the described microbial flocculant specifically comprises the following steps:

Preparation of A seed solution

The seed medium was prepared according to the following proportions: glucose, 10.0 g; K ₂ HPO ₄ , 5.0 g; MgSO ₄ ·7H ₂ O, 0.2 g; KH ₂ PO ₄ , 2.0 g; NaCl, 0.1 g; urea, 0.5 g; Yeast powder, 0.5g; distilled water 1L; pick the Enterobacter diazoae on the solid medium and inoculate it into the sterilized seed medium, culture it with aeration at 30±2°C for 24±6h to obtain the seed liquid;

B fermentation

Inoculate the seed liquid in the stable growth period obtained in step A into the fermentation medium at an inoculum size of 1-5%, culture in aeration at 30±2°C for 18±2h, centrifuge, remove the supernatant, and precipitate to obtain the microbial flocculant , the OD ₅₉₀ of the microbial flocculant is 2.0-2.4; the fermentation medium is prepared according to the following ratio: glucose, 8.0-12.0g; K ₂ HPO ₄ , 5.0g; MgSO ₄ ·7H ₂ O, 0.2g; KH ₂ PO ₄ , 2.0g; NaCl, 0.1g; sodium nitrate, 1.0-1.5g; distilled water 1L.

Further, in the preparation method of the microbial flocculant, the glucose in the seed medium in step A or the glucose in the fermentation medium described in step B can be replaced by any one of sucrose, lactose, mannitol and starch by equal weight. one or more; the sodium nitrate in the seed medium in step A or the sodium nitrate in the fermentation medium described in step B can be replaced by any one or more of peptone, urea, beef extract and ammonium sulfate in equal weight kind.

Further, in the preparation method, the biopreservation number of the Enterobacter diarrhoea strain is CCTCC M2013042.

The microbial flocculant obtained by the method.

Further, the microbial flocculant is bacterial precipitation.

Wherein, the microbial flocculant, the nucleotide sequence of the 16S rRNA of the Enterobacter diarrhoea strain is shown in SEQ ID NO:1.

The third object of the present invention is to provide a method for sedimentation of sludge in sewage, which is applicable to a wide range of objects and has obvious effects.

To achieve the above object, the technical solution of the present invention is:

Using the method of using the microbial flocculant to strengthen sludge precipitation in sewage treatment, the microbial flocculant is put into the sewage to be treated, and the ratio of the microbial flocculant to the sewage to be treated is 2%-10% by volume , processed for no less than 0.5 minutes.

Beneficial effects of the present invention: 1) In the pH range of 3-10, the flocculation rate of the microbial flocculant can reach more than 90%, with good pH stability; 2) In the temperature range of 20-70°C, the microbial flocculant There is no significant difference in the flocculation activity of the flocculant; 3) The flocculation time of the microbial flocculant is short, the efficiency is high, and there is no need to rely on metal ions as additives.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Fig. 1 is a morphological diagram of Enterobacter versicolor under the oil immersion microscope.

Figure 2 is a distribution diagram of different active ingredients.

Fig. 3 is the effect of different carbon sources in Fig. 3 on the flocculation activity of Enterobacter diazoea.

Fig. 4 is the effect of different nitrogen sources on the flocculation activity of Enterobacter diazoea.

Figure 5 shows the changes in the flocculation activity of Enterobacter dilatatus in different pH environments.

Fig. 6 Effects of different temperature pretreatments on the flocculation activity of Enterobacter diazoea.

Fig. 7 The change of the flocculation activity of Enterobacter dilatatus under different standing time.

Figure 8 is the effect of different dosages on the flocculation effect of Enterobacter diazoae.

Fig. 9 Effects of different metal ions on the flocculation effect of Enterobacter dilatatus.

Figure 10 is a picture of the application effect in sewage treatment, the test tube on the left is before treatment, and the test tube on the right is after treatment.

In the present invention, the Enterobacter gergoviae eth-2 (Enterobacter gergoviae eth-2) is sent to the China Typical Culture Collection Center (Wuhan University Collection Center) for preservation, the preservation number is CCTCC M2013042, and the address is located at Wuhan University, Wuhan, China, received The date is January 23, 2013.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, preferred embodiments of the present invention are described in detail below.

The amount of inoculum in the present invention takes volume as the unit of comparison, such as the ratio of the volume transferred into the seed solution to the volume of the culture solution after inoculation.

In the embodiment, the method for determining the flocculation rate is as follows: add 0.2 g of kaolin (4 g/L) to a 50 ml graduated cylinder, 1 ml of an _aqueous CaCl solution with a mass volume fraction of 1% (final concentration is 0.2 mg/ml), and 1 ml of the microorganism flocculant (OD ₅₉₀ value 2.0-2.4), then add water to 50ml, shake up and down 20 times, and let it stand for 5 minutes. At the same time, the kaolin suspension without microbial flocculant was used as a control. Take the supernatant at 2 cm, and measure the absorbance value at a wavelength of 550 nm with a spectrophotometer. The calculation formula of flocculation rate RF is: RF=(A－B)/A×100%. In the formula: A is the absorbance of the control supernatant; B is the absorbance of the sample supernatant.

Implementation Case 1 Screening and identification of high-efficiency flocculation bacteria

The flocculation strains were screened from activated sludge and landfill leachate, and the isolated strains were respectively inserted into 250ml wide-mouthed Erlenmeyer flasks equipped with 50ml of seed culture medium (see Example 3 below), and placed in a shaker at 30 Cultivate at 200rpm for 24 hours, and take the culture solution to measure the flocculation rate. A strain of Enterobacter urticaria with the highest flocculation activity was obtained, its biological deposit number was CCTCC M2013042 strain, and it was named eth-2. and use it as the target for subsequent experiments.

Identification method: As shown in Figure 1, eth-2 was sequenced by 16S rRNA (the sequencing result is shown in SEQ ID NO: 1) and compared in NCBI, and the similarity with Enterobacter.Gergoviae (AB682278.1) reached 99%.

The preparation of embodiment 2 culture medium

1. Culture medium preparation

1. Solid plate medium: tryptone, 10.0g; yeast powder, 5.0g; NaCl, 10.0g; agar powder, 20.0g; distilled water, 1L.

2. Seed medium: glucose, 10.0g; K ₂ HPO ₄ , 5.0g; MgSO ₄ 7H ₂ O, 0.2g; KH ₂ PO ₄ , 2.0g; NaCl, 0.1g; urea, 0.5g; yeast powder, 0.5g; distilled water 1L.

3. Fermentation medium: glucose, 10.0g; K ₂ HPO ₄ , 5.0g; MgSO ₄ ·7H ₂ O, 0.2g; KH ₂ PO ₄ , 2.0g; NaCl, 0.1g; sodium nitrate, 1.0g; distilled water 1L .

Second, the culture and fermentation conditions of microorganisms:

The culture conditions of the solid plate medium: the eth-2 strain was inoculated in the solid medium and cultured at 30°C for 1 day, and then stored in a refrigerator at 4°C after being matured.

The cultivation conditions of the seed medium: the seed medium was sterilized at 115°C for 20 minutes, and then inserted into the strains of the seed plate medium after sterilization, and cultivated in aeration at 30°C for about 24h.

The fermentation condition of the fermentation medium: the fermentation medium was sterilized at 115°C for 20 minutes, after sterilization, the strains were inoculated into the fermentation medium according to the inoculum size of 1%, and cultured with aeration at 30°C for about 18 hours.

3. Preparation of microbial flocculant:

Inoculate the eth-2 strain with the biological preservation number CCTCC M2013042 into the sterilized seed medium, take 1ml of the seed liquid and inoculate it into the fermentation medium after 24 hours, cultivate it at 30°C for 18 hours, and then centrifuge the fermentation liquid , remove the supernatant to get the bioflocculant. The colony of eth-2 is small, about 0.5mm, light yellow, opaque, round, with smooth edges and surface, wet and raised.

Implementation case 3eth-2 as preparation of microbial flocculant

1. Centrifuge a certain volume of culture medium at 8000rpm and 5minl to collect the supernatant and precipitate respectively, and wash the precipitate with distilled water for 3 times and then resuspend it in a considerable volume of distilled water. Compare the supernatant, the precipitate suspension and the fermentation broth respectively The flocculation effect on kaolin to determine the distribution of flocculation activity. The results are shown in Figure 2. The flocculation rates of the fermentation broth, bacterial suspension and supernatant were 90.45%, 94.36% and 63.20% respectively, indicating that the flocculation activity of eth-2 was mainly distributed in the bacterial sediment, so the following experiments In both experiments, bacterial pellets that were repeatedly washed with distilled water for 3 times and then resuspended in distilled water were used as microbial flocculants.

2. The effect of carbon source on the activity of microbial flocculant: select glucose, sucrose, lactose, mannitol, starch, citric acid, each 10g, as the carbon source of the fermentation medium, and the other components of the fermentation medium are: K2HPO4, 5.0g ; MgSO4·7H2O, 0.2g; KH2PO4, 2.0g; NaCl, 0.1g; urea, 0.5g; yeast powder, 0.5g; distilled water 1L. The effect of different carbon sources on flocculation activity was compared. The results are shown in Figure 3: eth-2 can utilize other carbon sources except citric acid, and the flocculation rates of utilizing glucose, sucrose, starch, lactose and mannitol are 86.17%, 78.03%, 55.67%, respectively. 86.31% and 81.48%. Because the cost of glucose is lower than that of lactose, and the difference in flocculation rate between the two is small, glucose was selected as the carbon source of the fermentation medium for subsequent experiments.

3. The effect of nitrogen source on the activity of microbial flocculant: select a single substance including peptone, urea, beef extract, sodium nitrate, ammonium sulfate, 0.1g each, and a mixture of 0.5g urea and 0.5g yeast powder as the fermentation medium Nitrogen source, other components of the fermentation medium are: glucose, 10g; K ₂ HPO ₄ , 5.0g; MgSO ₄ ·7H ₂ O, 0.2g; KH ₂ PO ₄ , 2.0g; NaCl, 0.1g; distilled water 1L. The effect of 1.0 g of different nitrogen sources on the flocculation activity was compared. The results (Figure 4) show that eth-2 can utilize all the above nitrogen sources, and the flocculation rates of tryptone, yeast powder, beef extract, urea, NaNO ₃ and (NH ₄ ) ₂ SO ₄ as nitrogen sources are 95.03% and 98.04% respectively. %, 53.63%, 92.40%, 98.00%, and 96.50%. Among them, the flocculation rate of using yeast powder and NaNO ₃ can reach more than 98%. Considering that the cost of NaNO ₃ is lower than that of yeast powder, and the difference in flocculation rate between the two is small, NaNO ₃ is selected as the nitrogen source of the fermentation medium for subsequent experiments. .

Implementation case 4 Stability of eth-2 microbial flocculants

1. The pH stability of eth-2 microbial flocculant

The microbial flocculant prepared in Example 3 was added to the kaolin suspension at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, and tested according to the above-mentioned flocculation rate determination method. The flocculation results (Fig. 5) show that in the pH range of 3-10, the flocculation rates of eth-2 are 95.80%, 90.28%, 93.63%, 91.85%, 94.10%, 93.97%, 92.32% and 94.70%, which can reach More than 90%, with good pH stability.

2. The temperature stability of eth-2 microbial flocculant

The microbial flocculants prepared in Example 3 were placed in water baths at 20°C, 30°C, 50°C, 70°C, and 90°C for 30 minutes, and tested according to the above-mentioned flocculation rate determination method. The flocculation results are shown in Figure 6, and the flocculation rates after treatment were respectively They were 87.89%, 90.02%, 82.58%, 82.63% and 60.69%. Except that the flocculation rate decreased significantly after 90℃ treatment, the 30min water bath treatment at 20℃, 30℃, 50℃ and 70℃ had little effect on the flocculation activity.

3. The effect of standing time on the activity of microbial flocculants

The microbial flocculant prepared in Example 3 was tested according to the above-mentioned flocculation rate determination method, the standing time was 0.5min, 2min, 5min, 10min and 15min respectively, and the kaolin suspension without microbial flocculant at the corresponding time point was tested. Absorbance was used as a control. The results (Figure 7) show that the flocculation rate of eth-2 can reach 97.82% when the action time is 0.5min, and the flocculation rates of 2min, 5min, 10min and 15min are 96.10%, 94.35%, 92.39% and 93.33% respectively, indicating that The flocculation time of eth-2 microbial flocculant is short, which helps to shorten the flocculation time in practical application.

4. Effect of the amount of microbial flocculant on the flocculation effect

The microbial flocculant prepared in Example 3 was tested for flocculation with different dosages of 0.05ml, 0.1ml, 0.5ml, 1.0ml and 2.0ml (OD ₅₉₀ is 2.0). The test results are shown in Figure 8. The flocculation rates of the above dosages are respectively 10.54%, 42.25%, 87.81%, 95.08%, and 94.61. The above results (Figure 8) show that a good flocculation effect can be obtained when the dosage is 0.5ml (OD ₅₉₀ is 2.0), so that the flocculation rate can reach more than 87%.

5. Effect of metal ion addition on microbial flocculant activity

Prepare the following solutions with a metal ion concentration of 0.09 mol/L: NaCl, KCl, MgCl ₂ , CaCl ₂ , AlCl ₃ and FeCl ₃ . Add 1ml of the above solutions to the graduated cylinder respectively, and then set a set of blanks without adding any ions, and test the microbial flocculant prepared in Example 3 according to the above-mentioned flocculation rate determination method. The test results are shown in Figure 9, the flocculation rates of NaCl, KCl, MgCl ₂ , CaCl ₂ , AlCl ₃ , FeCl ₃ and the blank are 94.31%, 94.83%, 95.94%, 97.35%, 66.88%, 20.16% and 95.13% respectively . The results (Fig. 9) indicated that the addition of metal ions did not significantly increase the flocculation rate of eth-2. Without the addition of metal ions, the flocculation rate of eth-2 can still reach more than 95%.

Implementation Case 5 Practical Application Test

Take 48ml of water before entering the CASS tank in the Luzhou Laojiao sewage treatment process and add it to a 50ml measuring cylinder, add 1ml of CaCl ₂ solution with a mass fraction of 1% and 1ml of -eth-2 suspension (OD ₅₉₀ is 2.4), up and down at room temperature Shake it upside down 20 times, let it settle for 5 minutes, and take the sewage without flocculant as the control. Take the supernatant at 2 cm, and measure the absorbance value at a wavelength of 550 nm with a spectrophotometer. It has been determined that the flocculation rate of eth-2 microbial flocculant to sewage can reach 90%.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims (8)

1. the application of Enterobacter.Gergoviae in the preparation of microbial flocculants, the biological deposit number of Enterobacter.Gergoviae is CCTCC M 2013042. 2. The preparation method of the microbial flocculant is characterized in that, specifically: fermenting and cultivating the Enterobacter diarrhoea bacterial classification with a medium suitable for Enterobacter diarrhoea, and separating, and the thalline or bacterium liquid is microbial Flocculant, the biological preservation number of the Enterobacter diarrhoea is CCTCC M 2013042. 3. the preparation method of microbial flocculant according to claim 2 is characterized in that, specifically comprises the following steps: Preparation of A seed solution The seed medium was prepared according to the following proportions: glucose, 10.0 g; K ₂ HPO ₄ , 5.0 g; MgSO ₄ ·7H ₂ O, 0.2 g; KH ₂ PO ₄ , 2.0 g; NaCl, 0.1 g; urea, 0.5 g; Yeast powder, 0.5g; distilled water 1L; pick the Enterobacter diazoae on the solid medium and inoculate it into the sterilized seed medium, culture it with aeration at 30±2°C for 24±6h to obtain the seed liquid; B fermentation Inoculate the seed liquid in the stable growth period obtained in step A into the fermentation medium at an inoculum size of 1-5%, culture in aeration at 30±2°C for 18±2h, centrifuge, remove the supernatant, and precipitate to obtain the microbial flocculant , the OD ₅₉₀ of the microbial flocculant is 2.0-2.4; the fermentation medium is prepared according to the following ratio: glucose, 8.0-12.0g; K ₂ HPO ₄ , 5.0g; MgSO ₄ ·7H ₂ O, 0.2g; KH ₂ PO ₄ , 2.0g; NaCl, 0.1g; sodium nitrate, 1.0-1.5g; distilled water 1L. 4. the preparation method of microbial flocculant according to claim 3 is characterized in that, the glucose in the fermented medium described in step B carries out equal weight and replaces any one or more in sucrose, lactose, mannitol and starch The sodium nitrate in the fermentation medium described in step B is replaced by any one or more of peptone, urea, beef extract and ammonium sulfate by equal weight. 5. the microorganism flocculant that the method described in claim 2 obtains. 6. The microbial flocculant according to claim 5, characterized in that, the microbial flocculant is bacterial precipitation. 7. microbial flocculant according to claim 5, is characterized in that, the nucleotide sequence of the 16S rRNA of described Enterobacter diaspora bacterial classification is as shown in SEQ ID NO:1. 8. Utilize the method for the sludge precipitation in the sewage treatment of the microbial flocculant strengthening described in claim 5, it is characterized in that, put described microbial flocculant in the sewage to be treated, the ratio of described microbial flocculant and sewage to be treated It is 2%-10% by volume, and the treatment is not less than 0.5 minutes.